Platelets are a component of blood that can aggregate when necessary for wound healing, for example. However testing platelet aggregation can reveal signs of a bleeding disorder or risk of thrombosis, or if a patient is not responding to anti-platelet therapy. Platelet aggregation tests can help diagnose problems with platelet function and determine whether the problem is due to one's genes, another disorder, or a side effect of medicine.
Platelets are known to aggregate under a variety of conditions and in the presence of a number of different reagents. “Platelet aggregation” is a term used to denote the adherence of one platelet to another. When they aggregate, platelets change from a discoid shape to a more spherical form, extend long processes known as pseudopodia and become sticky. As a result, the platelets stick to one another and to the damaged tissue, thus plugging gaps or holes in the blood vessel wall. Although the primary response of platelets is to aggregate, a secondary release reaction may also occur, during which platelets release materials which accelerate the clotting process.
Platelets' ability or inability to respond to particular aggregating reagents is the basis for differentiating platelet dysfunction from normal, for example.
Aggregation can be induced in a sample by adding aggregating agents to platelet-rich plasma or whole blood. Platelet aggregation depends on the presence of calcium, fibrinogen and one or more plasmatic factors, and an aggregating agent. Platelet aggregation will vary with different aggregating agents and with their concentration.
One method of testing platelet aggregation is optical aggregometry. For example, in 1962, Born described the aggregation of platelets by ADP and modified a colorimeter to monitor continuously this aggregation in platelet rich plasma. These modifications included incubation at 37° C., stirring and recording the change in light transmission over time on a pen recorder. This method is commonly referred to as Light Transmission Aggregometry (LTA).
With LTA, platelets are in a suspension of plasma isolated from an anticoagulated blood sample by a relatively low centrifugal force centrifugation. This material is known as platelet-rich plasma (PRP). Platelet-poor plasma (PPP) is prepared by centrifuging the blood sample at a relatively high force.
The sample chamber or chambers in multiple channel instruments are heated to 37° C. Provision is made for stirring of the sample because platelet to platelet contact is necessary to the determination of in vitro platelet aggregation. A beam of light shines through the sample cuvette. Photodiodes detect the light able to pass through the sample. A sample cuvette containing PRP is measured and a sample cuvette containing PPP is measured. PRP is arbitrarily considered to be 0% light transmission or 0% aggregation; PPP is considered to be 100% light transmission or 100% aggregation. The difference in light transmission outputs from the photodiodes is transferred to recording devices.
When an agonist or aggregating agent is added to the cuvette containing PRP and the platelets respond, changes in light transmission occur and are recorded over time by the recording device.
When the platelets undergo shape change in response to an agonist, their larger size allows less light to pass through the PRP: this is recorded as less light transmission through the sample relative to the PPP. If the dose of aggregating agent is strong enough to cause the platelets to adhere to each other and form aggregates, more light is able to pass through the PRP sample. The change in light transmission recorded, over time, shows a trend towards the platelet poor plasma, or 100% light transmission.
As is well known, in-vitro aggregation recordings are characterized by their appearances:                shape change        a first wave of aggregation (primary aggregation) that may reverse and return towards the PRP baseline        Irreversible second wave aggregation that occurs when the platelets' secreted granule contents become the stimulus and cause additional aggregation.        
Aggregation curves are also characterized by:                the maximum amount of change in light transmission caused by the agonist (percent aggregation)        the slope- or rate-of the aggregation, in % change of aggregation per minute.        
Multiple aggregating agents and dosages are usually used to stimulate the platelets. Different aggregating agents stimulate different pathways of activation in the platelets: either binding sites or metabolic pathways. Different concentrations of agonists are used to elicit a family of curves (dose response curves).
The pattern of responses to these test panels is compared to established normal response patterns and established abnormal response patterns. This information is considered to relate to the platelet function component of homeostasis.
In 1980, Cardinal and Flower described an impedance method for measuring aggregation in whole blood (U.S. Pat. No. 4,319,194). In their method, a very small electric current is passed between two electrodes. During initial contact with the blood the electrodes become coated with a monolayer of platelets. When an agonist is added, platelets aggregate on the monolayer increasing the impedance. This increase in impedance is recorded on a pen recorder.
In the impedance method, platelets are tested in anti-coagulated blood, without the need to isolate them from other components of blood. Because there is no need to centrifuge the specimen to produce an optically transparent suspension of cells, the entire platelet population is tested. The process of testing consumes less technical time; and labile factors in the blood itself that may influence platelet function are preserved.
A typical impedance aggregometer consist of a sample chamber or chambers (in multiple channel instruments,) heated to 37° C. The device typically includes apparatus to stir the samples, commonly utilizing non-magnetic disposable stir bars. Cuvettes containing the test sample and a stir bar are placed in the chamber(s).
The impedance (or electrical resistance) method of aggregation is non-optical. An electrode probe assembly is inserted into a cuvette containing a test sample. The electrode probe assembly consists basically of two precious metal wires that are immersed in the sample. An AC voltage in the millivolt range is applied to the probe circuit. The instrument measures the electrical resistance or impedance between the two immersed wires.
During a brief period of equilibration, a monolayer of platelets forms on the exposed portions of the wires, resulting in a stable impedance value. This stable baseline of impedance is assigned a value of zero ohms of resistance. An agonist is added to the cuvette and the stimulated platelets aggregate to the platelet monolayer on the immersed wires. This accumulation of platelets adds electrical resistance to the circuit. The changes in resistance are measured and quantified in ohms (the measurement of electrical resistance). The impedence measurement of the aggregated sample is generally run continuously for four to six minutes after the addition of an agonist.
Results of impedance aggregation tests are quantified by:                Ohms of aggregation at a given time in the test        Slope, or rate of the reaction, in ohms change per minute        Maximum extent of aggregation, in ohms.        
The increase in impedance is directly proportional to the mass of the platelet aggregate. Impedance aggregation in blood is more sensitive to the aggregating effects of ristocetin so it may be more sensitive to von Willebrand disease than the bleeding time or vWF (ristocetin co-factor) assay. Impedance aggregation in blood is not dependent on the optical characteristics of the sample, so tests can be performed on lipemic and thrombocytopenic samples. As centrifugation is not required, impedance aggregation is especially useful in conditions where megathrombocyte count is increased.
The impedance method allows the study of platelets in the more physiologically representative whole blood environment. Sample preparation is greatly simplified, and preserves labile modulators such as prostacyclin and thromboxane A2, resulting in a testing environment proven to be more sensitive to the effects of many anti-platelet drugs (e.g., aspirin, dipyridamole, abciximab, clopidogrel, ticagrelor, ticlopidine, prasugrel, etc. . . . ).
In 1984, Freilich developed a low cost disposable electrode for measuring impedance aggregation by substituting for the wire electrodes conductive ink printed on a plastic nonreactive base (U.S. Pat. No. 4,591,793). This device is less expensive than the Cardinal device and is disposable after each test; however, there are disadvantages to the Freilich device. The platelets have difficulty adhering to the exposed conductive surface of the Freilich device, probably due to the surface being thin. Sometimes the aggregated platelets break off the surface, causing a sudden change in impedance. Although the Freilich device is inexpensive to manufacture, the measurements returned by the device can be inconsistent and not reproducible.
In 1997 Freilich et al. developed an improved low cost disposable electrode that overcame the reproducibility problems of their prior device (see, U.S. Pat. No. 6,004,818). The inventors discovered that the most reproducible configuration has the electrodes side-by-side with respect to the flow pattern. This configuration allows the platelets to stick to the face and the area between the electrodes, facilitating the formation of a bridge of platelets between the electrodes, which results in a stronger bond of platelets to the electrodes.
This device consists of two metal plates with a connection tab at one end and tip at the other end. The two plates are separated by an electrical insulator comprised any non-conducting material, such as mylar, plastic or teflon, which will separate the electrodes by the proper amount. Except for the tips, the plates are isolated from the sample by a non-conductive coating comprised of any insulating material, such as plastic or epoxy, which is non-reactive with the blood sample. The electrode tips are side by side with respect to the flow pattern. The tips are non-circular in cross-section, preferably rectangular, and most preferably square. The advantages of square tips are that at least one planar face of one electrode tip is adjacent and parallel to at least one planar face of the opposing electrode tip. Also, the square electrode tips are easier to produce than round electrode tips because a stamping process can be used to make the electrode out of flat metal to form an electrode plate.
The position fixing means are either a pair of molded plastic, semi-circular fins extending outwardly from the molded plastic coating the electrodes or molded plastic parts with slots for the placement of the electrodes. It is of considerable importance to keep the electrode tips in the proper placement in the cuvette so it is necessary to have these elaborate means to hold the electrode assembly in place.
There are several disadvantages to this device, which prevented it from ever going to market. First although it is less expensive than the Cardinal and Flower device, it is still too expensive to be disposable in today's cost conscious laboratory. Additionally, this device is difficult to manufacture which would result in a high rejection rate. Therefore this device has never been produced nor sold.
However, a variation of this device was produced and sold. This device used Square electrode pins held in the optimal position in the sample. The main difference is instead of plates the pins were attached to a kapton strip that had electrical circuit lines on it These circuit lines electrically connected the pins to tabs at the top of the a kapton strip. These tabs are used as contacts for connecting to the electrical circuit of the instrument.
This design works well in a 1 mL sample, however, when it was adapted it to a smaller sample, there was a spontaneous reaction of the platelets most likely due to a sheer force in the smaller sample cuvette. This spontaneous reaction makes this device unusable in a small sample cuvette. The small sample size is more beneficial because less patient blood is needed to run the assay.
Because the prior design failed when it was adapted to fit a smaller sample, there is a need for a workable electrode for smaller blood samples. This electrode needs to be easy to use and low cost and have the ability to test platelet function in whole blood in a small sample size.
The prior design was used in a 1 mL sample size, in a large cuvette (0.44″×1.83″). With the new device, the sample size will be from 250 μL to 300 μL. This smaller sample size requires a smaller sample cuvette with provision to stir the sample at 1000 RPM, the traditional stirring speed for platelet aggregation.